BMC Structural Biology
BioMed Central
Open Access
Methodology article
(NZ)CH...O Contacts assist crystallization of a ParB-like nuclease
Neil Shaw1, Chongyun Cheng1, Wolfram Tempel2, Jessie Chang2, Joseph Ng3,
Xin-Yu Wang1, Sarah Perrett1, John Rose2, Zihe Rao1,4, Bi-Cheng Wang2 and
Zhi-Jie Liu*1,2
Address: 1National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing, 100101, China, 2Southeast
Collaboratory for Structural Genomics, Department of Biochemistry and Molecular Biology, University of Georgia, GA 30602, USA, 3Laboratory
of Structural Biology and Department of Biological Sciences, University of Alabama in Huntsville, Huntsville, AL 35899, USA and 4Laboratory of
Structural Biology, Tsinghua University, 100084, China
Email: Neil Shaw - neilshaw@moon.ibp.ac.cn; Chongyun Cheng - ccy@moon.ibp.ac.cn; Wolfram Tempel - tempel@gmail.com;
Jessie Chang - Jessie@secsg.uga.edu; Joseph Ng - ngj@email.uah.edu; Xin-Yu Wang - xinyuwang_cn@yahoo.com.cn;
Sarah Perrett - sperrett@ibp.ac.cn; John Rose - rose@bcl4.bmb.uga.edu; Zihe Rao - raozh@xtal.tsinghua.edu.cn; BiCheng Wang - wang@bcl1.bmb.uga.edu; Zhi-Jie Liu* - zjliu@ibp.ac.cn
* Corresponding author
Published: 7 July 2007
BMC Structural Biology 2007, 7:46
doi:10.1186/1472-6807-7-46
Received: 10 February 2007
Accepted: 7 July 2007
This article is available from: http://www.biomedcentral.com/1472-6807/7/46
© 2007 Shaw et al; licensee BioMed Central Ltd.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0),
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Abstract
Background: The major bottleneck for determination of 3 D structures of proteins using X-rays
is the production of diffraction quality crystals. Often proteins are subjected to chemical
modification to improve the chances of crystallization
Results: Here, we report the successful crystallization of a nuclease employing a reductive
methylation protocol. The key to crystallization was the successful introduction of 44 new cohesive
(NZ) CH...O contacts (3.2 – 3.7 Å) by the addition of 2 methyl groups to the side chain amine
nitrogen (NZ) of 9 lysine residues of the nuclease. The new contacts dramatically altered the
crystallization properties of the protein, resulting in crystals that diffracted to 1.2 Å resolution.
Analytical ultracentrifugation analysis and thermodynamics results revealed a more compact
protein structure with better solvent exclusion of buried Trp residues in the folded state of the
methylated protein, assisting crystallization.
Conclusion: In this study, introduction of novel cohesive (NZ)CH...O contacts by reductive
methylation resulted in the crystallization of a protein that had previously resisted crystallization in
spite of extensive purification and crystallization space screening. Introduction of (NZ)CH...O
contacts could provide a solution to crystallization problems for a broad range of protein targets.
Background
The resolution and accuracy of the structural information
provided by X-rays is unsurpassed when compared to
other techniques employed to resolve the 3 D structure of
proteins [1]. However, not all proteins can be made available for X-ray diffraction studies because of the inherent
difficulty in obtaining single crystals of adequate size and
quality [2]. A subset of such proteins that resist crystallization can be salvaged by employing a reductive methylation protocol [3-11]. A 26 kD nuclease from Pyrococcus
furiosus used in the current study had resisted crystallization inspite of extensive purification and crystallization
Page 1 of 12
(page number not for citation purposes)
BMC Structural Biology 2007, 7:46
space screening. A number of techniques have been developed to improve a given target's potential for crystallization. Truncation of disordered regions [12,13],
mutagenesis of surface residues [14-16] and chemical
modification of proteins [3] have been proven effective in
this regard and a number of protein structures have been
solved successfully. We decided to modify the surface
lysines of the nuclease by reductive methylation in an
attempt to crystallize the protein. The rationale behind
targeting the amine nitrogen (NZ) of the nuclease arose
from a number of considerations. Lysines harbouring free
NZ atoms almost always reside on the surface of protein
molecules [17]. The thermodynamic cost for ordering the
highly flexible solvent exposed side chains of lysines is
exorbitant [18]. Since crystallization is a surface phenomenon, a disordered lysine side chain has a profound negative impact on the formation of stable, uniform, inter
molecular contacts essential for packaging of protein molecules in a crystal lattice. Interestingly, we found that
methylation of surface lysines resulted in a decrease in the
free energy of folding of the protein. This is consistent
with the entropic cost of reduced flexibility in the native
state due to formation of new intra molecular interactions, which in turn will lower the barrier to crystallization. We further probed the effect of methylation at the
molecular level and show that cohesive (NZ)CH...O
bonds assisted crystallization of the nuclease.
Results
Generation of intra molecular contacts
After purifying the protein to homogeneity, two methyl
groups were covalently linked to the free amine nitrogen
of lysine residues [3-11] (Figure 1A) by treating the protein with formaldehyde and dimethylamine borane complex (DMAB). Electron density for only 9 amine nitrogens
out of 32 (including the N-terminal amine group), could
be unambiguously assigned as dimethylated (Figure 1B).
The methyl carbon is protonated because of the strong
electron withdrawing nature of the NZ atom. The protonated methyl carbon can form ionic interactions with carbonyl and carboxyl oxygens of surrounding residues. The
(NZ)CH...O bond formed by the CH2 group of MLY159
and the carboxyl oxygen OE2 of Glu156 is shown in Figure 1C. The length of the cohesive interaction is 3.27 Å.
Forty new intra molecular (NZ)CH...O contacts in the
range 3.2 to 4.0 Å were generated because of the methylation, of which 25 interactions were between 3.2 to 3.8 Å
(see Additional file 1). The large number of cohesive intra
molecular contacts generated helps immobilize the flexible regions of the protein molecules (Figure 1D), which is
crucial for the formation of stable intermolecular contacts
and may lower the entropic cost of crystallization. The
temperature factor of the MLY159NZ was 12.18. The addition of methyl groups to the amine nitrogen and the for-
http://www.biomedcentral.com/1472-6807/7/46
mation of cohesive bonds through the protonated carbon
of the methyl group seemed to have significantly lowered
the B factor of the NZ atom of MLY159 indicating a localized side chain. Glu216 forms two strong (NZ)CH...O
cohesive bonds with the methyl groups of MLY159 (Figure 2A). The B factors of the OE1 and OE2 atoms of
Glu216 involved in the formation of the hydrogen bond
were 14.3 and 12.36 respectively. The low B factors demonstrate immobilization of the Glu216 side chain possibly because of the new (NZ)CH...O bonds. Similarly, the
B factors of both the carboxyl oxygens of Glu156 participating in (NZ)CH...O interactions were also low. B factors
of 16.64 for the OE1 oxygen and 20.24 for the OE2 oxygen indicate the side chain of Glu156 to be severely
restricted in movement. The B factors, however, need to
be interpreted cautiously in absence of a structure of the
unmodified protein for comparison.
Generation of inter molecular contacts
The methyl carbons of all the dimethylated lysine residues
were involved in the formation of multiple new symmetry-generated intermolecular contacts. The synthesis of the
intermolecular contacts was initiated by gradual evaporation of the solvent in presence of different chemicals.
Commercially available sparse matrix screens under oil
were used for screening the best chemical environment for
formation of intermolecular bonds [19,20]. The structure
reveals 96 new symmetry-generated inter molecular contacts in the range 3.2 – 5.0 Å involving the (NZ)CH group
and 28 of these inter molecular contacts are of the
(NZ)CH...O type and in the range 3.2 – 4.0 Å (see Additional file 2). A significant number of these interactions
were within the optimal range of 3.2 – 3.7 Å for CH...O
hydrogen bonds [21]. The intermolecular contacts involving the methyl groups of MLY112 and MLY201 are shown
in Figures 2B and 2C respectively. The B factors for the NZ
atoms of MLY112 and MLY201 were 25 and 15 respectively. The new intra and inter molecular contacts formed
by the covalently linked methyl groups with the surrounding residues seem to have lowered the B factor values of
the NZ atom indicating a localized side chain. This helps
the packaging of the molecules in the crystal and the formation of a compact crystalline lattice.
Although the contacts formed were predominantly
(NZ)CH...O bonds, a few (NZ)CH...N bonds were also
formed (Figure 3A and 3B). The side chain nitrogens of
Arg35 interact with the CH1 methyl carbon of MLY172.
Water molecules surrounding these interactions possibly
help disperse the excess positive charge on the nitrogens
by bridging ionic interactions [22]. The NH1 nitrogen of
Arg35 forms a hydrogen bond with water 119, which is
hydrogen bonded to the carboxylic oxygens, OD1 and
OD2, of Asp 39. Similarly, the NH2 nitrogen of Arg35
forms a hydrogen bond with water 117, which is hydro-
Page 2 of 12
(page number not for citation purposes)
BMC Structural Biology 2007, 7:46
A
http://www.biomedcentral.com/1472-6807/7/46
B
C
D
Figure of
Nature
1 (NZ)CH...O contacts of the methylated nuclease
Nature of (NZ)CH...O contacts of the methylated nuclease. A. Diagrammatic representation of the (NZ)CH...O
bond. The side chain amine nitrogen (NZ) of lysine residues (blue circle) polarizes the covalently linked methyl carbon (green
circle). The polarized methyl carbon acts like a proton donor and forms ionic interactions with neighbouring carboxyl oxygens
(red circle). The optimal range for the (NZ)CH...O bond distance is between 3.2 – 3.7 Å. The angle of the approach of a proton towards the lone pair of electrons is generally between 90 – 180°. In order to calculate the angle, the position of hydrogen
(grey circle) for X-ray structures is usually deduced. B. Electron density for the dimethylated lysine MLY159. The 2 | Fo | – | Fc
| electron density map was contoured at 1.5 σ. C. The protonated methyl carbon, CH2, of MLY159 is seen forming a 3.6 Å
(NZ)CH...O bond with the carboxyl oxygen of E156. The amino acids are represented as sticks. The (NZ)CH...O bond is
shown as a dashed magenta line. D. A cartoon representation of the modified protein showing the dimethylated lysines
engaged in the formation of numerous intra molecular (NZ)CH...O contacts. Such contacts localize side chains and loops in
space, resulting in a compact protein molecule. (NZ)CH...O bonds are shown as dashed blue lines.
Page 3 of 12
(page number not for citation purposes)
BMC Structural Biology 2007, 7:46
http://www.biomedcentral.com/1472-6807/7/46
Figure 2 of intra and inter molecular (NZ)CH...O contacts for the nuclease
Generation
Generation of intra and inter molecular (NZ)CH...O contacts for the nuclease. A. The intra molecular (NZ)CHO
contacts generated for MLY159 as a result of the chemical modification of the nuclease are shown as dashed magenta lines. B,
C. The symmetry generated inter molecular contacts synthesized for MLY112 and MLY201 during the crystallization of the
nuclease are shown as magenta dashed lines. Residues involved in the formation of (NZ)CH...O bonds are represented as
sticks while water molecules are represented as spheres.
Page 4 of 12
(page number not for citation purposes)
BMC Structural Biology 2007, 7:46
gen bonded to the carbonyl oxygen of Gln170. The excess
positive charge on the Arg35 nitrogens is relayed to the
carboxyl oxygens of Asp35 and carbonyl oxygen of
Gln170 via water molecules (data not shown). Similarly,
in case of Gln31, the excess positive charge is relayed to
the side chain OE1 oxygen atom.
In addition, a significant number of water molecules were
observed to form (NZ)CH...O contacts (Figure 3C).
Analytical ultracentrifugation
The 26 kD protein was subjected to analytical ultracentrifugation analysis in order to determine the effect of the
reductive methylation protocol on the purity, aggregation
state and shape of the protein. The sedimentation velocity
experiment results revealed that both non-methylated and
methylated proteins were pure, homogenous and monomeric (Figure 4). However, a qualitative decrease in the
diffusion co-efficient (D) could be observed for the methylated protein (Figure 4B). This is consistent with
decreased flexibility of side chains and a more compact
structure in the methylated protein.
Thermodynamic stability of nuclease before and after
modification
In order to further investigate the mechanism by which
chemical modification of the protein affected the ease of
crystallization, we performed equilibrium denaturation
experiments using heat or the chaotropic agent guanidinium chloride (GdmCl) as denaturants. The nuclease contains three Trp residues, of which two (W74 and W102)
are buried in the folded structure, making intrinsic fluorescence a sensitive probe of global structural changes. We
also used far-UV circular dichroism (CD), which monitors
changes in secondary structure. Both proteins were found
to be extremely thermostable and no secondary structural
changes were detected below 90°C (not shown). However, we found that the proteins could be completely
unfolded in 4 M GdmCl at 25°C (Figure 5A). Further,
thermal denaturation could be achieved in the presence of
a non-denaturing concentration of GdmCl (Figure 5B).
Interestingly, we found that the chemical modification
resulted in a decrease in the mid-point of unfolding in
both GdmCl and thermal denaturation (Figures 5A, B),
which corresponds to a decrease in the free energy of
unfolding, Δ GU,H20, of 18 ± 3 kJ/mol (Table 1). Surface
lysines are likely to be involved in salt-bridges or other
favourable interactions that may be lost on methylation,
resulting in a decrease in stability. Another explanation is
that there is a decrease in entropy in the folded state of the
modified protein due to formation of intra molecular
(NZ)CH...O interactions involving the introduced methyl
groups, which reduces the flexibility of the protein. Solvent effects may also contribute to the observed difference
in stability, for example, greater ordering of water mole-
http://www.biomedcentral.com/1472-6807/7/46
cules around hydrophobic methyl groups at the protein
surface could also lead to a greater loss in entropy on folding for the methylated protein. We also observed that the
λmax of the fluorescence spectrum for the native state was
blue-shifted for the modified protein, whereas the λmax for
the denatured states was similar (Figure 5A). This is consistent with a more compact structure and better solvent
exclusion of buried Trp residues in the folded state of the
chemically modified protein. Reduced entropy of the solvated folded structure, together with the formation of
additional favourable interactions in the crystalline state,
may account for the greater ease of crystallization after the
chemical modification
Discussion
Structure determination holds the key to unravelling the
mechanisms by which proteins drive the machinery of all
living organisms for survival and propagation. In spite of
several dramatic technological advances in automation
and information science, the key step for successful crystallographic structure determination – production of high
quality crystals – continues to remain a resource intensive
bottleneck [1]. A first step towards addressing this bottleneck would be to identify the variables involved in crystallization. The nature of the protein is the single most
important factor that influences crystallization. From a
crystallization point of view – size, charge, hydrophobicity, hydrophilicity, flexibility, oxidation state and posttranslational modifications like phosphorylation, glycosylation and myristylation – define the nature of a protein. Since the nature of the protein often varies with
function, there is no universal crystallization strategy that
would work for all proteins. To maximize the chances of
crystallization it is crucial to identify and target an
attribute of the protein that would have a profound effect
on the crystallization. Surface lysines offer one such target.
Lysines almost always reside on the surface of proteins.
The solvent exposed side chains of lysines are highly
mobile and prevent the formation of inter molecular contacts essential for the assembly of a crystalline lattice [18].
Defects observed in protein crystals like low resolution
and twinning is also a manifestation of the flexibility of
the side chains found on the surface of the protein.
Locking the side chain amine nitrogen of lysines with the
electron negative carboxyl oxygens of glutamic and aspartic acid side chains via cohesive ionic interactions will
result in the immobilization of these side chains (Figure
2). Although the presence of CH...O bonds has been demonstrated in proteins, nucleic acids and carbohydrates
[21-27], the (NZ)CH...O bonds introduced in the current
study have never been described before. Methyl groups are
very effective in mediating and bridging the physical distances between the free amine nitrogen and the side chain
carboxyl oxygens for the formation of cohesive ionic inter-
Page 5 of 12
(page number not for citation purposes)
BMC Structural Biology 2007, 7:46
http://www.biomedcentral.com/1472-6807/7/46
Figure 3 contacts and water molecules in (NZ)CHO bonds observed for the methylated nuclease
(NZ)CHN
(NZ)CHN contacts and water molecules in (NZ)CHO bonds observed for the methylated nuclease. A, B. The
side chain nitrogens of R35 and Q31 are seen forming (NZ)CHN bonds with the methyl groups of MLY172 and MLY159
respectively. Excess positive charge on the nitrogen is dispersed via ionic interactions mediated by water molecules [22] or as
in case of Q31, relayed directly to electron negative oxygen OE1. C. A number of water molecules are seen participating in the
formation of (NZ)CHO bonds. Residues involved in the formation of (NZ)CHN and (NZ)CH...O bonds are represented as
sticks while water molecules are represented as spheres.
Page 6 of 12
(page number not for citation purposes)
BMC Structural Biology 2007, 7:46
http://www.biomedcentral.com/1472-6807/7/46
Figure 4 ultracentrifugation analysis of methylated and non-methylated protein
Analytical
Analytical ultracentrifugation analysis of methylated and non-methylated protein. The methylated (A) and nonmethylated (B) protein were subjected to analytical ultracentrifugation analysis as described in Methods. The sedimentation
velocity experiments showed the protein remained pure and monomeric after the chemical modification. There was an obvious decrease in the diffusion co-efficient for the protein after the methylation indicating a more compact shape.
Page 7 of 12
(page number not for citation purposes)
BMC Structural Biology 2007, 7:46
http://www.biomedcentral.com/1472-6807/7/46
Figure
Comparison
5 of thermodynamic stability of modified and non-modified protein
Comparison of thermodynamic stability of modified and non-modified protein. A. GdmCl-induced equilibrium
denaturation of the nuclease (squares) and its modified variant (circles) in 20 mM phosphate buffer pH 7.4 at 25°C. The protein
concentration was 1 μM. The fit to a two-state model is shown and the parameters obtained are displayed in Table 1. B. Thermally-induced equilibrium denaturation of the nuclease (squares) and its modified variant (circles) in 20 mM phosphate buffer
pH 7.4 in the presence of 2.5 M GdmCl. The protein concentration was 25 μM. The fit to a two-state model is shown and the
free energy value obtained after extrapolation to standard conditions (25°C in the absence of GdmCl) is the same within error
as that obtained by GdmCl denaturation (Table 1).
actions. Methyl groups can be covalently linked to the free
amine nitrogen (NZ) with very high specificity using formaldehyde as the methyl group donor in the presence of
dimethylamine borane complex [3]. The amine nitrogen
sitting adjacent to the methyl carbon, (NZ)CH, has an
inductive effect on the methyl carbon resulting in a highly
polarized carbon, which can act as a proton donor. The
negative charge required for the electro neutrality is compensated by the lone pair of electrons of carboxyl oxygens
found on the side chains of neighbouring glutamic and
aspartic acid residues. The net effect is a cohesive ionic
(NZ)CH...O interaction between the side chains leading
to a compact, rigid protein molecule with localized side
chains and loops. The positive charge on the methyl carbon can also be dispersed indirectly via the participation
of water molecules (Figure 3C).
The maximum and minimum distances for a cohesive
CH...O bond have been set at 3.2 Å and 3.7 Å respectively,
depending on the direction of the approach of the proton
towards the lone pair of electrons of oxygen [21]. Deviations in the above distances are frequently observed
Table 1: Comparison of thermodynamic parameters for ParB before and after modification§
Protein
[GdmCl]1/2
(M)
m
(kJ/mol/M)
ΔGU,H2O
(kJ/mol)
Nuclease
Nuclease (modified)
3.01 ± 0.01
2.74 ± 0.01
19 ± 1
14.4 ± 0.5
57.4 ± 3.0
39.4 ± 1.4
§ The values for GdmCl denaturation were obtained by fitting data to a standard 2-state model [29] where [GdmCl]
1/2 is the midpoint and m the
slope of the denaturation transition. ΔGU,H2O is the free energy of unfolding at 25°C in the absence of denaturant. The errors shown are the
standard errors from the fit. The dependence of ΔGU on the GdmCl concentration is given by ΔG(GdmCl) = Δ Gu-m [GdmCl]. Conditions were as
described in the legend for Figure 5A. The thermal denaturation data (Figure 5B) was also fitted to a 2-state model and was extrapolation to
standard conditions (25°C in the absence of denaturant) as described previously [30]. The value of ΔGU,H2O thus obtained is the same within the
error as that shown above.
Page 8 of 12
(page number not for citation purposes)
BMC Structural Biology 2007, 7:46
http://www.biomedcentral.com/1472-6807/7/46
owing to the steric interactions between the atoms
involved in the formation of the (NZ)CH...O bonds and
the surrounding residues. In the present study, except for
a couple of contacts involving MLY221, the minimum distance of all the CH...O bonds was 3.2 Å. The only short
contacts – a 3.0 Å link between the CH2 methyl carbon of
MLY221 and a phosphate oxygen, and a 3.1 Å bond
between the carboxyl oxygens of E36 and the MLY221
methyl carbons, resulted in the distortion of the conformation of the MLY221 and the glutamic acid (Figure 6),
suggesting steric clashes due to the excessive closeness of
the methyl carbon and the oxygen atoms. Thus, 3.2 Å
seems to be the minimum van der Waals limit for all
(NZ)CH...O interactions. Detailed analysis of the electron
density map showed no other obvious changes to any
other amino acid confirming the specificity of the modification.
The methylated protein lost its ability to cleave DNA
(results not shown). The dimethylation of K221 sitting at
the edge of the active site may sterically hinder access of
the active site to the incoming DNA. It is also possible that
an overall conformational change in the protein induced
by the chemical modification affects the catalytic site and
compromises the function of the protein. However, such
loss-of-function due to methylation has not been reported
previously. Further studies are warranted in order to determine the exact cause for the inactivation of the methylated
protein.
When a protein is set up for crystallization, the molecules
are moving randomly in search of compatible bonding
partners. Some of the inter molecular contacts generated
during the course of random collisions are sustained. As
more and more solvent evaporates, a number of these
interactions become permanent. Eventually it leads to one
of two possible outcomes. If the inter molecular bonding
is heterogeneous, as in case of protein molecules that
assume more than one conformation due to the presence
of unstructured domains, flexible loops and side chains,
or presence of partially unfolded regions, this will result in
a disordered protein aggregate commonly referred to as
precipitate. Poorly diffracting or defective crystals are also
a consequence of the conformational flexibility of protein
molecules. A homogenous inter molecular bonding pattern between protein molecules, as in the case of structurally rigid molecules, results in optimal packing of the
molecules in a crystal lattice. A direct manifestation of the
chemical modification of the protein is the reduction in
number of degrees of freedom available to the protein for
assuming different conformations. Introduction of
(NZ)CH...O bonds curbs the movement of side chains
and fixes their orientation in space. This produces uniform bonding partners and decreases the steric clashes
between molecules during the packing of the lattice. The
Figure 6 of MLY221
Occupancy
Occupancy of MLY221. MLY221 shows 2 conformations.
Conformation A has occupancy of 65%. The distance of the
oxygen atoms of E36 and the PO- 4 ligand from the (NZ)CH
group of the MLY221 is shorter than the permissible limit of
3.2 Å for (NZ)CHO bonds, resulting in the distorting of
MLY221 and E36. The 2 | Fo | – | Fc | electron density map
was contoured at 1.5 σ
observed reduction in the free energy of folding for the
modified protein is consistent with reduced flexibility
leading to lower entropy in the native state; ordering of
water molecules around the surface-exposed methyl
groups may also contribute to this. Crystallization will be
favoured for the modified protein by the reduction in
entropy of the solvated structure and the involvement of
the methyl groups in inter molecular (NZ)CH...O interactions upon crystallization
A pre-requisite to the success of the crystallization strategy
described here is the requirement of a highly pure homogenous protein sample [28] usually obtained by a combination of different chromatography steps (Figure 7).
Presence of homologous and heterologous impurities can
compromise the effectiveness of the chemical modification of the protein.
Conclusion
In conclusion, introduction of (NZ)CH...O bonds by
reductive methylation of surface lysines as a means to salvage targets is simple, fast, economical and non laborious.
It could be the first method of choice for rescuing a target
before attempting a more extensive approach involving
mutagenesis.
Page 9 of 12
(page number not for citation purposes)
BMC Structural Biology 2007, 7:46
http://www.biomedcentral.com/1472-6807/7/46
Figure 7flow sheet of the crystallization strategy for the nuclease
Process
Process flow sheet of the crystallization strategy for the nuclease. The protein sample is purified to homogeneity
using a combination of chromatographic methods. The pure and homogenous protein is chemically modified in order to localize the side chains and loops. The resultant compact protein molecule is then screened against a variety of chemical environments using commercially available sparse matrix screens to determine the best condition for the self-assembly of the protein
molecules into a crystalline lattice.
Methods
Protein production and purification
The 26 kD ParB nuclease was expressed with a N-terminal
hexa Histidine tag. The gene was PCR amplified from the
genomic DNA of Pyrococcus furiosus and cloned into pET28a vector (Invitrogen). E coli BL21 cells containing the
plasmid were grown in LB cultures. The protein was purified using affinity chromatography followed by size exclusion chromatography. Nucleic acids were removed by
hydroxyapatite chromatography (GE Healthcare). Further
purification was achieved by an ion exchange step. The
protein was exchanged into crystallization buffer (20 mM
Tris, pH8.0, 200 mM NaCl) using a size exclusion column.
Chemical modification
Methylation of the protein was done as described before
[3] using formaldehyde and dimethylamine-borane complex (DMAB). In brief, 10 mg/ml of protein in a 1.5 ml
eppendorf tube covered with aluminium foil was mixed
with 40 μl of 1 M solution of formaldehyde (Sigma) and
20 μl of 1 M solution of DMAB (Sigma) in the dark at 4°C.
The reaction mixture was incubated under shaking conditions for 2 h, after which the chemical additions were
repeated. Finally, 10 μl of DMAB was added and the reaction mixture was incubated overnight. Excess chemicals
were removed by size exclusion chromatography.
Crystallization
Methylated and non-methylated protein was set up for
crystallization under oil as described before [19,20]. 1-μl
crystallization drops contained 0.5 μl protein mixed with
Page 10 of 12
(page number not for citation purposes)
BMC Structural Biology 2007, 7:46
0.5 μl of crystallization solution. Commercially available
sparse matrix screens (Hampton Research, Molecular
Dimensions) were used for crystallization screening. Crystals for structure determination were produced using a
precipitant solution consisting of 600 mM sodium dihydrogen phosphate, 2.4 M di-potassium hydrogen phosphate, 200 mM sodium chloride, 100 mM HEPES, p H
7.3.
Data collection and structure determination
Data collection and structure determination will be
described elsewhere.
http://www.biomedcentral.com/1472-6807/7/46
Authors' contributions
NS, JC, and JN produced, purified, and crystallized the
protein. NS and CC did the chemical modification and
analytical ultracentrifugation analysis. X-YW and SP carried out the thermodynamic characterization of the protein. Z-JL, WT, and JR solved the structure and did the
analysis. Z-JL designed the experiments and interpreted
the data. B-CW and ZR conceived the study and participated in its design and co-ordination. NS, SP, and Z-JL
drafted the manuscript. All authors read and approved the
final manuscript.
Accession numbers
Analytical Ultracentrifugation
Analytical sedimentation velocity experiments were carried out using a ProteomeLab™ XL-I protein characterization system (Beckman Coulter). An-60Ti rotor was used to
centrifuge a 10 mg/ml protein sample suspended in 50
mM phosphate buffer, 150 mM NaCl, pH 7.2, at 60,000
rpm. Absorbance was read at 280 nm. A set of 93 scans
were collected at 1 min intervals. Data was analyzed using
Sedfit software
Equilibrium denaturation experiments
All fluorescence denaturation experiments were performed in 20 mM phosphate buffer pH 7.4 at 25°C, with
a final protein concentration of 1.2 μM. Samples of modified and unmodified protein were mixed with different
concentrations of GdmCl and allowed to equilibrate overnight before measurements were taken. Refolding experiments were also performed, by denaturing the protein for
8 h in 6 M GdmCl, then diluting the protein to give different final concentrations of GdmCl as for unfolding experiments. The intrinsic fluorescence spectra were recorded
between 300 and 400 nm after excitation at 280 nm in a
Hitachi F-4500 spectrofluorimeter. The fluorescence data
were plotted as the centre of spectral mass as described
previously [29]. GdmCl denaturation was found to be
reversible and the data were fitted to a 2-state model [30].
Far-UV CD experiments were performed on a Pi-star 180
instrument (Applied Photophysics, UK) using a cell of 1
mm optical path length and the same buffer as for fluorescence experiments. The protein concentration was 25 μM.
The temperature was changed at a rate of 1°C per 10 min,
with a step size of 0.5°C, for both heating and cooling.
Thermal denaturation was found to be reversible and the
data were analyzed as described previously [31]
List of abbreviations used
MLY – Methylated lysine
Coordinates for the structure reported in the current study
have been deposited in the Protein Data Bank under
accession code 1VK1
Additional material
Additional file 1
Intra molecular contacts$ of the (NZ)CH group of methylated lysines
of the nuclease. The data presented in the table is a list of intra molecular
contacts generated during the methylation of the nuclease.
Click here for file
[http://www.biomedcentral.com/content/supplementary/14726807-7-46-S1.doc]
Additional file 2
Symmetry generated contacts* of the (NZ)CH group of the methylated lysines of the nuclease. The data presented in the table is a list of
intra molecular contacts generated during the methylation of the nuclease.
Click here for file
[http://www.biomedcentral.com/content/supplementary/14726807-7-46-S2.doc]
Acknowledgements
This work was funded by the 863 (Grant 2006AA02A316) and 973 (Grants
2006CB910901, 2006CB910903, 2006CB500703) Projects of the Ministry
of Science and Technology of China, the National Natural Science Foundation of China (Grants 30470363, 30670427), the National Institutes of
Health (Grant 1P50 GM62407), the University of Georgia Research Foundation, the Georgia Research Alliance. Crystallographic data was collected
at Southeast Regional Collaborative Access Team (SER-CAT) 22-ID beamline at the Advanced Photon Source, Argonne National Laboratory. Use of
the Advanced Photon Source was supported by the U. S. Department of
Energy, Office of Science, Office of Basic Energy Sciences, under contract
number W-31-109-Eng-38.
References
1.
2.
3.
(NZ) – Side chain amine nitrogen
Stevens R, Wilson I: Industrializing structural biology. Science
2001, 293:519-520.
Dale G, Oefner C, D'Arcy A: The protein as a variable in protein
crystallization. J Struct Biol 2003, 142:88-97.
Rayment I: Reductive alkylation of lysine residues to alter crystallization properties of proteins. Methods in Enzymology 1997,
276:171-179.
Page 11 of 12
(page number not for citation purposes)
BMC Structural Biology 2007, 7:46
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
21.
22.
23.
24.
25.
Geoghegan K, Cabacungan J, Dixon H, Feeney R: Alternative
reducing agents for reductive methylation of amino groups
in proteins. Int J Pept Protein Res 1981, 17:345-352.
Gerken T, Jentoft J, Jentoft N, Dearborn D: Intramolecular interactions of amino groups in 13C reductively methylated hen
egg-white lysozyme. J Biol Chem 1982, 257:2894-2900.
Means G, Feeney R: Reductive alkylation of amino groups in
proteins. Biochemistry 1968, 7:2192-2201.
Rypniewski W, Holden H, Rayment I: Structural consequences of
reductive methylation of lysine residues in hen egg white lysozyme: An X-ray analysis at 1.8Å resolution. Biochemistry 1993,
32:9851-9858.
Schubot F, Waugh D: A pivotal role for reductive methylation
in the de novo crystallization of a ternary complex composed
of Yersinia pestis virulence factors YopN, SycN and YscB. Acta
Cryst 2004, D60:1981-1986.
Kobayashi M, Kubota M, Matsuura Y: Crystallization and
improvement of crystal quality for X-ray diffraction of maltooligosyl trehalose synthase by reductive methylation of
lysine residues. Acta Cryst 1999, 55(Pt 4):931-933.
Walter T, Meier C, Assenberg R, Au K-F, Ren J, Verma A, Nettleship
J, Owens R, Stuart D, Grimes J: Lysine methylation as a routine
rescue strategy for protein crystallization. Structure 2006,
14:1617-1622.
Liu Z-J, Tempel W, Ng J, Lin D, Shah A, Chen L, Horanyi P, Habel J,
Kataeva I, Xu H, Yang H, Chang J, Huang L, Chang S-H, Zhou W, Lee
D, Praissman J, Zhang H, Newton G, Rose J, Richardson J, Richardson
D, Wang B-C: The high-throughput protein-to-structure pipeline at SECSG. Acta Crystallogr 2005, 61(Pt 6):679-684.
Dale G, Kostrewa D, Gsell B, Stieger M, D'Arcy A: Crystal engineering: deletion mutagenesis of the 24 kDa fragment of the
DNA gyrase B subunit from Staphylococcus aureus. Acta Crystallogr D Biol Crystallogr 1999, 55:1626-1629.
Kwong P, Wyatt R, Robinson J, Sweet R, Sodroski J, Hendrickson W:
Structure of an HIV gp120 envelope glycoprotein in complex
with the CD4 receptor and a neutralizing human antibody.
Nature 1998, 393:648-659.
Derewenda Z: Rational protein crystallization by mutational
surface engineering. Structure 2004, 12:529-535.
Longenecker K, Garrard S, Sheffield P, Derewenda Z: Protein crystallization by rational mutagenesis of surfaceresidues: Lys to
Ala mutations promote crystallization of RhoGDI. Acta Crytallogr D Biol Crytallogr 2001, 57(Pt 5):679-688.
Mateja A, Devedjiev Y, Krowarsch D, Longenecker K, Dauter Z,
Otlewski J, Derewenda Z: The impact of Glu→Ala and
Glu→Asp mutations on the crystallization properties of
RhoGDI: the structure of RhoGDI at 1.3 Å resolution. Acta
Crytallogr D Biol Crytallogr 2002, 58(Pt 12):1983-1991.
Baud F, Karlin S: Measures of residue density in protein structures. Proc Natl Acad Sci USA 1999, 96:12494-12499.
Avbelj F, Fele L: Role of main-chain electrostatics, hydrophobic
effect and side-chain conformational entropy in determining
the secondary structure of proteins.
J Mol Biol 1998,
279:665-684.
Chayen N, Shaw Stewart P, Maeder D, Blow D: An automated system for micro-batch protein crystallization and screening. J
Appl Cryst 1990, 23:297-302.
D'Arcy A, Elmore C, Stihle M, Johnston J: A novel approach to
crystallising proteins under oil.
J Crystal Growth 1996,
168:175-80.
Derewenda Z, Lee L, Derewenda U: The occurrence of C-HO
hydrogen bonds in proteins. J Mol Biol 1995, 252:248-262.
Magalhaes A, Maigret B, Hoflack J, Gomes J, Scheraga H: Contribution of unusual arginine-arginine short-range interactions to
stabilization and recognition in proteins. J Protein Chem 1994,
13:195-215.
Kump R, Damewood J: Do nitromethane and molononitrile
form C-H...O hydrogen bonds? Implications for molecular
recognition by crown ethers. J Chem Soc Chem Commun
1988:621-622.
Taylor R, Kennard O: Crystallographic evidence for the existence of C-H...O, C-H...N and C-H...Cl hydrogen bonds. J Amer
Chem Soc 1982, 104:5063-5070.
Wahl M, Sundaralingam M: CH...O hydrogen bonding in biology.
Trends Biochem Sci 1997, 22:97-102.
http://www.biomedcentral.com/1472-6807/7/46
26.
27.
28.
29.
30.
31.
32.
Desiraju G: The C-H...O hydrogen bond in crystals: What is it?
Acc Chem Res 1991, 24:290-296.
Donati A, Ristori S, Bonechi C, Panza L, Martini G, Rossi C: Evidences of strong C-HO bond in an o-carboranyl beta-lactoside in solution. J Am Chem Soc 2002, 124:8778-8779.
McPherson A: Introduction to Protein Crystallography. Methods: A Companion to Methods in Enzymology 2004, 34:254-265.
Lima L, Zingali R, Foguel D, Monteiro R: New insights into conformational and functional stability of human alpha-thrombin
probed by high hydrostatic pressure. Eur J Biochem 2004,
271:3580-3587.
Maxwell K, Wildes , Zarrine-Afsar A, De Los Rios M, Brown A, Friel
C, Hedberg L, Horng J-C, Bona D, Miller E, Vallée-Bélisle A, Main E,
Bemporad F, Qiu L, Teilum K, Vu N-D, Edwards A, Ruczinski I,
Poulsen F, Kragelund B, Michnick S, Chiti F, Bai Y, Hagen S, Serrano
L, Oliveberg M, Raleigh D, Wittung-Stafshede P, Radford S, Jackson S,
Sosnick T, Marqusee S, Davidson A, Plaxco K: Protein folding:
Defining a "standard" set of experimental conditions and a
preliminary kinetic data set of two-state proteins. Protein Science 2005, 14:602-616.
Su X, Sanbo Q, Xian-Ming P: Thermal and conformational stability of Ssh10b protein from archaeon Sulfolobus shibattae.
Biochem J 2004, 382:433-440.
Potterton E, Briggs P, Turkenburg M, Dodson E: A graphical user
interface to the CCP 4 program suite. Acta Cryst 2003, 59(Pt
7):1131-1137.
Publish with Bio Med Central and every
scientist can read your work free of charge
"BioMed Central will be the most significant development for
disseminating the results of biomedical researc h in our lifetime."
Sir Paul Nurse, Cancer Research UK
Your research papers will be:
available free of charge to the entire biomedical community
peer reviewed and published immediately upon acceptance
cited in PubMed and archived on PubMed Central
yours — you keep the copyright
BioMedcentral
Submit your manuscript here:
http://www.biomedcentral.com/info/publishing_adv.asp
Page 12 of 12
(page number not for citation purposes)